Human Hypertension caused by Mutations in WNK Kinases Science Paper Wilson et al 2001

Why is the study of Mendelian Forms of Hypertension important? Hypertension affects 25% of most adult population and is a common risk factor for common causes of morbidity and mortality Molecular Pathogenesis of common forms of hypertension is poorly understood Studying rare Mendelian form of hypetension provide clues to pathways Identify new drug targets and approaches to treatment

Features of PHAII/Gordons Syndrome Autosomal Dominant Hypertension; ↑ renal salt reabsorption Hyperkalemia; ↓ renal K+ excretion Metabolic acidosis; ↓ renal H+ secretion These features suggest a defect in RENAL ELECTROLYTE HANDLING. Treatment – Thiazide diuretics. Autosomal Dominant – You just need one copy of the mutated gene to have the phenotype Hypertension – high blood pressure Hyperkalemia – increased Potassium levels in the blood But with - Normal glomerular filtration - Normal aldosterone secretion Acidosis – increased acidity of body fluids Thiazide diuretics – act by inhibiting salt reabsorption in the distal nephron.

Genetics of Affected Families Linkage ↓ Genotype - (Look for mutations) (Size of fragment) PCR Sequencing Southern blotting Deletions Missense mutations

Genetics of Affected Families – K22 Genome wide scan shows linkage to Chromosome 12 PHA II Kindreds (affected have typical features of PHAII) Null allele D12s94 3 Additional marker close to D12s94 PCR: - using primers usually 42 kb apart in WT, - only 600bp in affected individuals confirm deletion and 41kb fragment in exon 1 of WNK1 Typical features of PHAII Blood pressure > 140/90 mm Hg Hyperkaleamia, Normal Glomerular filtration rate, Suppressed plama renin activity, Normal or elevated aldosterone levels, Hyperchloremia… First found Linkage Linkage to most telomeric 2cM Centimorgan of Chr 12p Then Additional markers in telomeric region Genotyping – Affected members looked homozygotes But the allele passed for affected parents to offspring violated mendelian transmission. (Eg D12S94) In all 4 informative matings (4 zeros), offspring inherited no allele from affected parent Findings indicate null allele Consistent with deletion within the disease linked choromosome segment

Genetics of affected families – K4 Null alleles at STS45K STS60K PCR Novel 24Kb product Absent in WT DNA sequencing Deletion of 21Kb NB – this deletion is located within the deletion shown in the K22 family. Compare WNK 1 expression levels – 5x increase in WNK1 expression compared to unaffected individuals Expression studies in K4 and WT show there I s 5 fold increase in WNK1 expression is affected individuals

Families with WNK 1 paralog mutations Kindreds K13, K23, K11, K21 Paralogs of WNK1 ↓ ↓ ↓ WNK2 WNK3 WNK4 Chr9 Chr X Chr 17 Additional PHAII loci have previously been mapped to Chromosome 1 and Chromosome 17 (Mansfield et al 1997) 4 missense mutations in WNK4 found in PHAII kindreds. But what about families that did not have WNK1 mutations Looked for WNK1 paralogs. Paralogs are genes related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one. Mutations found in highly conserved domain

Causes of PHAII No mutation in WNK 1 or WNK 4 Affected Families Chromosome 12 WNK 1 mutation Chromosome 17 WNK 4 mutation Chromosome 1 ? Deletion Missense mutations Genetic gain of function 41kb deletion Intron 1 21kb deletion Intron 1 a.a 565 Gln -> Glu Exon 7 a.a 564 Asp -> Ala Exon 7 a.a 562 Glu -> Lys Exon 7 a.a 1185 Arg -> Cys Exon 7 K22 K4 K13 K23 K11 K21

Expression of WNK 1 and 4 WNK 1 10kb transcript-kidney Add expression in leukocytes WNK 1 10kb transcript-kidney 12kb transcript- predominantly in heart and skeletal muscle WNK 4 Approx 4.4kb expressed exclusively in kidney

Localisation WNK 1 WNK 4 WNK 4 WNK 1 Results not shown Anti WNK4-RED Distal Convoluted Tubule Staining Anti WNK4 -RED Anti NCCT- GREEN Distal Convoluted Tubule Staining Anti WNK1-RED Anti NCCT- GREEN Renal Cortex Staining Anti WNK1-RED Anti AQP2- GREEN Renal Cortex Staining Anti WNK4-RED Anti AQP2- GREEN Fluorescent Microscopy Results – Co-localisation of Anti WNK1 and Anti AQP2 shows WNK1 is found in the connecting tubule and collecting duct Anti NCCT – GREEN – Marker of Distal Convoluted Tubule Results – Co localisation of Anti WNK1 and anti NCCT WNK1 – Present in cytoplasm WNK4 – Present exclusively in intercellular junctions in DCT - Present in cytoplasm in CCD Results not shown Anti WNK4-RED ZO-1 GREEN – A tight junction complex Results show that WNK is found in the intercellular junctions Anti AQP2- GREEN- Marker of connecting tubule and collecting duct Anti NCCT- GREEN- Marker of DCT

Summary Mutations in WNK1 & WNK4 cause PHAII leads to a genetic gain of function mechanism. Clustering of mutations in highly conserved domain suggest disruption at this site normally required for normal regulation. Both kinases are found in the distal nephron which is well known to play a key role in electrolyte homeostasis, This process is disrupted in PHAII individuals. Identification of upstream and downstream targets will help to understand WNK signalling pathways. In DCT, salt reabsorption is mediated by NCCT In CD, Na+ is reabsorbed by ENaC

WNK 4 Missense mutations WNK1 in vitro Prevents WNK4 interacting with NCCT WNK 4 Missense mutations NCCT │ ↑overactivity ↑net sodium Reabsorption ↑plasma volume ↑cardiac output ↑ BP Hypertension NCCT In vitro WNK4 interacts with NCCT and Loses its ability to suppress it Hyperkaleamia WNK4 directly Interacts with K+ channel ROMK